In this proposal we attempt to develop experimental approaches for the isolation, identification and cell culture of tracheal epithelial and mesenchymal cell subpopulations. The sources of cells will be hamster and baboon tracheas. We intend to utilize a variety of reagents to discriminate among and isolate different airway cells using laser flow cytometry (LFC) and differential bacterial adherence. In conjunction with multiparameter LFC, the reagents to be used include lectins, and monoclonal and polyclonal antibodies to cell surface components (markers) such as fibronectin, keratin and laminin. In one instance, monoclonal antibody and its complementary bacterial antigen believed to be involved in the attachment of pathogen (Mycoplasma pneumoniae) to respiratory cells will be used to separate cells by LFC. The separation of respiratory cells will also be attempted using fixed, bacterial monolayers of organisms known to possess binding affinities for eukaryotic cells. Various immunocytochemical electron microscopic techniques (e.g., Staphylococcus Protein A-colloidal gold, lectin-colloidal gold) will be used for ultrastructural examination of freshly isolated and passaged cells to aid in their identification and assessment of purity. Further long term goals include the cell culture of isolated or enriched cell populations obtained by the above methods and their biochemical study in vitro. Recent successes in the culture of respiratory epithelial cells attest to the feasibility of such studies. The isolation and biochemical analysis of specific repiratory cell populations is just in its infancy and may significantly enhance our understanding of pathological conditions such as chronic bronchitis, cystic fibrosis and asthma.